The invention is related to the area of genetic alterations of antigens as potent vaccines. In particular it is related to the field of mutagenesis.
The use of vaccines to build immunity against foreign and/or endogenous polypeptides provides an effective and selective strategy for treating the underlying cause of disease. In particular is the use of killed viruses such as the polio mellitus and the Hepatitis B virus (John T. J. (2000) New Engl. J. Med. 14:806-807). Standard methods for generating vaccines against candidate pathogenic organisms or molecules are known by those skilled in the art. Vaccines for human use are developed in animal models to survey for the ability of killed or defective whole agents such as parasites, viruses or recombinant polypeptides to cause immunity against infection of the pathogenic agent (Boyce T. G. et al. (2000) Vaccine 19:217-226). Briefly, rodents such as mice or rats are injected with a purified antigen in the presence of adjuvant to generate an immune response (Boyce T. G. et al. (2000) Vaccine 19:217-226). Unfortunately, not all antigens are capable of eliciting a strong immune response when injected into a host organism (Hoshino Y. and A. Z. Kapikian (2000) J. Health Popul. Nutr. 18:5-14; Orenstein W.A. et al. (2000) Am. J. Public Health 90:1521-525; Lechmann M. and T. J. Liang (2000) Semin. Liver Dis. 20:211-226). While the reasons for the lack of immune response are not clear, some factors, such as the lack of T-cell epitopes which are important for stimulating cellular-mediated immune responses, may be absent within a given antigen (Ausiello C. M. et al. (1999) Infect. Immun.67:4064-4071; Brosstoff S. (1995) Adv. Exp. Med. Biol. 383:249-254). In the case of parasitic infections, the development of effective vaccines has been hampered by the presence of many different developmental stages that occur within an infected host and that a diverse array of allelic forms occurs within genes encoding for prominent surface antigens (MALARIA OBSTACLES AND OPPORTUNITIES, Oaks, S. C. et al., Eds., National Academy Press, p 1, 1991; Anders, R. F. xe2x80x9cVaccines Against Asexual Blood Stages of Plasmodium falciparumxe2x80x9d NEW GENERATION VACCINES, 2nd Ed., Anders, R. F., pp. 1035-1055, 1997). It is believed by many skilled in the art that the generation of highly antigenic polypeptides may overcome these limitations and produce a protective immune response to pathogens (McLeod R. et al. (1995) Curr. Opin. Immunol.7:539-552).
A method for generating diverse sequences within a polypeptide would be useful for the creation of more potent therapeutic agents. Moreover, the generation of randomly altered nucleotides and encoded polypeptide residues throughout an entire antigen molecule may result in new reagents that are: 1) more antigenic; 2) more immunogenic; and 3) have beneficial pharmacokinetic properties.
The invention described herein is directed to the use of random genetic mutation of a polypeptide in vivo by blocking the endogenous mismatch repair (MMR) activity of a host cell yielding structurally altered antigens that can be screened for antigenicity and immunogenicity in comparison to the wild type molecule. The use of mammalian cell-based high throughput screens as taught by this application will facilitate identification of randomly altered antigens that may serve as effective vaccines. Moreover, the invention describes methods for repeated in vivo genetic alterations and selection for antigens with enhanced immunogenicity and pharmacokinetic profiles.
The ability to develop and screen genetically altered mammalian cells that secrete structurally altered polypeptides in a high throughput manner provides a valuable method for creating vaccines for therapeutic development. A potential problem in generating potent vaccine antigens against endogenous to the mammalian host is the source of antigen production. In many instances recombinant polypeptides that are naturally produced by mammalian cells are generated recombinantly using insect, yeast or bacterial expression systems. These sources typically produce large amounts of proteins that are distinct from the mammalian-produced polypeptides, and may differ from the natural protein due to altered folding or altered post-translational modifications such as hyperglycosylation. The invention described herein is directed to the creation of genetically altered mammalian cell hosts that produce structurally altered polypeptides as vaccine agents via the blockade of MMR.
The present invention facilitates the generation of highly antigenic polypeptides as vaccines. The advantages of the present invention are further described in the examples and figures described herein.
The present invention provides methods for generating genetically altered antigens in vivo, whereby the antigen possesses desired biochemical property(s), such as, but not limited to, increased antigenicity and immunogenicity. One method for identifying antigens with increased antigenicity is through the screening of mismatch repair (xe2x80x9cMMRxe2x80x9d) defective cell clones that produce desired antigens.
The invention also provides methods for rendering cells expressing a target antigen hypermutable. The cells include, but are not limited to rodent, primate, human, plant, yeast or bacterial cells. The antigens can be generated from endogenous genes or from introduced transgenes.
The invention also provides methods for generating genetically altered cell lines that express antigenic polypeptides.
In some embodiments, the invention provides methods for generating genetically altered cell lines that produce immunogenic polypeptides.
In other embodiments, the invention provides methods for producing an antigen expression cassette for high throughput screening of altered polypeptides in vivo.
In other embodiments, the invention provides methods of mutating a gene of interest in a mismatch repair defective cell.
In some embodiments, the invention provides methods of creating genetically altered antigens in vivo by blocking the MMR activity of the cell host.
Still other embodiments of the invention provide methods of creating genetically altered polypeptides in vivo by transfecting genes encoding for an antigen in a MMR defective cell host.
The invention also embraces methods of creating antigens with increased immunogencity due to genetic alterations within the antigen-encoding gene by blocking endogenous MMR of the cell host.
In some embodiments, the invention provides methods of creating a library of randomly altered antigens from mammalian cells by blockade of MMR of the cell host.
In other embodiments, the invention provides methods of creating antigens with enhanced pharmacokinetic profiles due to genetic changes within the encoding gene by blocking endogenous MMR of the cell host.
The invention also provides methods of creating genetically altered antigens in MMR defective cells as vaccine agents.
In some embodiments, the invention provides methods for high throughput screening of antigens produced by MMR defective cells.
These and other objects of the invention are provided by one or more of the embodiments described below. In one embodiment of the invention, a method for making MMR defective cell lines expressing a target antigen will be provided. A polynucleotide encoding a dominant negative allele of an MMR gene is introduced into a target antigen-producing cell. The cell becomes hypermutable as a result of the introduction of the gene.
In another embodiment of the invention, an isolated hypermutable cell producing antigenic peptides is provided. The cell is defective for mismatch repair and exhibits an enhanced rate of hypermutation. The cell produces a polypeptide from a mutated gene encoding for the polypeptide.
In another embodiment of the invention, a method is provided for introducing a mutation into an endogenous gene encoding for a target polypeptide. A polynucleotide encoding a dominant negative allele of a MMR gene is introduced into a cell. The cell becomes hypermutable as a result of the introduction and expression of the MMR gene allele. The cell further comprises a gene of interest. The cell is grown and tested to determine whether the gene encoding for a polypeptide of interest harbors a mutation.
In another embodiment of the invention, a method is provided for producing a cell-based screening assay to identify antigenic proteins as vaccines. A polynucleotide encoding a dominant negative allele of a MMR gene is introduced into a cell expressing a secreted antigen. The cell becomes hypermutable as a result of the introduction of the gene. The cell is grown and conditioned medium from the cell is tested for the expression of antigenic polypeptides.
In another embodiment of the invention, a gene, or set of genes encoding for polypeptides or a combination therein, are introduced into a mammalian cell host that is defective in MMR. The cell is grown and clones are analyzed for antigens with enhanced antigenicity.
In another embodiment of the invention, a method is provided for producing a cell-based screening assay to identify antigenic proteins as vaccines. A polynucleotide encoding a secreted antigen is introduced into a naturally MMR defective cell. The gene is hypermutable as a result of the introduction of MMR deficiency. The cell is grown and conditioned medium from the cell is tested for the expression of antigenic polypeptides.
In another embodiment of the invention, a method will be provided for restoring genetic stability in a cell containing a polynucleotide encoding for a dominant negative allele of a MMR gene. The expression of the dominant negative MMR gene is suppressed and the cell restores its genetic stability including but not limited to genetic stability within the antigen-encoding genes.
In another embodiment of the invention, a method will be provided for restoring genetic stability in a cell containing a polynucleotide encoding a dominant negative allele of an MMR gene and a newly selected phenotype. The expression of the dominant negative mismatch repair gene is suppressed and the cell restores its genetic stability and the new phenotype is stable.
These and other embodiments of the invention provide the art with methods that can generate enhanced mutability in cells and animals as well as providing cells and animals harboring potentially useful mutations for the large-scale production of highly antigenic polypeptides as potent vaccines.